The major structural proteins in epithelial cells are keratins, which occur as part of an intermediate filament (IF) scaffold in their cytoplasm. The IF scaffold provides a mechanical resilience that is vital to epithelial cells in the face of mechanical trauma, and accordingly mutations in keratin genes underlie many inherited epithelial fragility syndromes. Two Types of keratin proteins, I and II, are encoded by >40 genes in mammalian genomes. This dual character reflects a strict requirement for co-assembly, and underlies the co-regulation of Type I and Type II genes by epithelial cells. The observed pairwise and differentiation-specific expression of most Type I and type II keratin genes suggests a role for these proteins in promoting aspects of the cytoarchitecture and function of epithelial cells. This project will study two Type I keratins, K16 and K17. These keratins are of interest to us because of their regulation during development, their distribution in mature skin appendages (hair, nail, glands), and their induction in wound edge tissue after skin injury and in the context of skin diseases (psoriasis, carcinoma). The application posits that expression of K16 and K17 promotes a plastic cytoarchitecture enabling keratinocytes to migrate and differentiate along multiple pathways. To define the specific function(s) of K16 and K17, there are plans to characterize mouse strains carrying null mutations in the corresponding genes. The applicants recently reported that targeted expression of K16 can only partially compensate for the skin blistering phenotype produced by a null mutation in the K14 gene, a related Type I keratin. They also showed that the functional differences between K14 and K16 are encoded in their carboxy-terminal portion, and that the latter contributes to the cytoplasmic organization and mechanical properties of Ifs. There are also plans to characterize the mechanical properties of the three major types of keratin co-polymers found in the soft epithelia of the skin: K5-K14, Kl-K10, and K6-K161K17. Through mutagenesis studies will be used to define the biochemical basis for these properties, and their regulation. These studies will help us understand the role(s) of keratin proteins in the skin.